1. Field of the Invention
The present invention generally relates to enzyme assays and more specifically to a method and device for a dry format sialidase assay for the diagnosis of infectious diseases.
2. Discussion of the Related Art
The present invention discloses methods and devices for the detection of sialidase activity in fluid samples and in particular for the detection of sialidase activity in bodily fluids for the diagnosis of infectious diseases.
Sialidases, also known as neuraminidases, are the enzymes that catalyze the cleavage of terminal sialic residues from carbohydrate moieties of glycoproteins, glycolipids, and proteoglycans. Sialidases specific for varying ketosidic linkages perform many biological functions. They are found in viruses, bacteria, parasites, and vertebrates, including mammals. Sialidases are associated with many diseases. Bacterial and viral sialidases may act as pathogenic factors in microbial infections by processing carbohydrate moieties of glycoproteins and glycolipids on the host cell surfaces. Elevated sialidase activities in bodily fluids have been shown to be associated with bacterial and viral infectious diseases including bacterial vaginosis and influenza. In humans, abnormal production of sialidases is associated with diseases such as sialidosis and increased Pseudomonas aeruginosa infection in cystic fibrosis patients.
In particular, high levels of sialidase activity have been found in women having bacterial vaginosis, an abnormal condition of the vaginal ecosystem caused by a flora shift from the lactobacillus species present in normal conditions to overgrowth of a variety of aerobic and anaerobic vaginal bacteria, including Gardnerella vaginalis, Bacteroides, Prevotella, Mobilincus and Mycoplasma species. Bacterial Vaginosis (BV) is a common disease in reproductive-age women and is responsible for approximately one-third of all cases of Vulvovaginitis. BV is associated with gynecologic and obstetric complications including an increased risk for salpingitis, endometritis, pelvic inflammatory disease (PID), chorioamnionitis, premature rupture of membrane, and pre-term birth. BV is also associated with higher susceptibility to sexually transmitted pathogens, including HIV. A number of studies have shown BV to be associated with elevated sialidase activity. Other studies have shown that direct sialidase assay on vaginal fluid may identify the BV syndrome. Correlation was also found between elevated sialidase activities and an increased risk of preterm birth and low weight infants, suggesting that a sialidase activity test may predict adverse pregnancy outcome in women diagnosed with BV. The correlation found between elevated sialidases and pre-term birth also suggests that sialidase might be responsible for this complication by causing degradation of the protective mucus gel. The higher susceptibility to sexually transmitted pathogens might also be associated with the enzymatic degradation of the mucus gel that otherwise helps protect against such pathogens.
Conventional methods for diagnosing BV rely on an expert assessment and laboratory tests. Currently, there are two standard methods for diagnosing BV, the Amsel clinical criteria test and the Gram stain Nugent score. The Amsel criteria require the presence of at least three of four clinical signs set forth by Amsel et al. (Amsel et. al. Am. J. Med. 74:14-22, 1983). The alternative, more objective, method is to use a Gram stained evaluation of vaginal smears with the Nugent criteria or score (Nugent et al. J Clin Microbiol 29:297-301, 1991). This method scores the smears in a standardized manner by quantification of some of the cell types present designated as Lactobacillus, Gardnerella vaginalis, Bacteroides and Mobilincus ‘morphotypes’. The relative proportions of bacterial morphotypes give a score between 0 and 10. A score of ≦3 is normal, 4-6 is intermediate and ≧7 is BV positive. However, complete evaluation of BV by the Amsel criteria or by Gram-stained smears is time consuming. Moreover, it requires skilled personnel and microscopic capabilities, which are not available in many of the physician offices or other clinic settings attended by women with BV. As a result, samples need to be sent to a laboratory and results are further delayed, or more often, laboratory tests are not performed and diagnosis is based on clinical signs only, which may be misleading. There is therefore a need for a rapid point-of-care diagnostic test for BV which will aid in quick diagnosing BV while the patients are still in clinic and will allow starting appropriate treatment with no delay.
Recently, a new point-of-care BV test, distributed under the name BVBlue®, has been developed. The BVBlue® is a two-step liquid-phase chromogenic test, based on the detection of increased sialidase activity in vaginal fluid. The test kit contains a solution of a sialidase substrate, referred to as IBX-4041 which when exposed to bacterial sialidase undergoes chemical reaction to yield sialic acid and a compound referred to as IBX-4050. The test procedure involves the immersion of vaginal swab in the IBX-4041 solution to let it stand for 10 minutes. A drop of a 1M NaOH is then added to generate a blue or green color upon a positive result and a yellow color when the result is negative. The BVBlue test was evaluated by a number of studies and has been shown to be a useful point-of-care diagnostic tool for BV, exhibiting good sensitivity and specificity as compared with Amsel criteria and Nugent score (Myzuik et al., J. Clin. Microbiol., 41:1925, 2003), Bradshaw et al, J. Clin. Microbiol., 43:1304, 2005). While the BVBlue® provides a liquid phase sialidase point-of-care test, there is still a continuous need for other simple point-of-care tests for BV detection, and in particular for a one-step dry format sialidase assays, preferably of the strip format type.
Accordingly, it is the general object of the present invention to provide a simple, one-step, dry format assay for the detection of sialidase activity in a fluid sample.
More specifically it is the object of the invention to provide a rapid point-of-care test for the diagnosis of infectious disease associated with sialidase activity
It is a further object of the present invention to provide a test as set above for the diagnosis of bacterial vaginosis.
A further object of the invention is to provide a test as set above in a lateral flow format for facilitating analyte concentration and signal enhancement.
It will be appreciated that while the above and following description focuses on BV detection, the present invention is not limited to the detection of BV sialidases. Rather, the method and devices of the invention may be applied for the detection of sialidases of any other origin including bacterial, viral, protozoa or human origin and for the diagnosis of other infectious sialidase-related diseases, including influenza, T. cruzi infection and Pseudomonas aeruginosa infection, as well as for human sialidase-associated disorders such as sialidosis. For example the disclosed invention may be used as a first screen for the detection of influenza virus.